When human diploid fibroblasts in culture are deprived of serum, they enter a non-proliferating, resting state. Upon readdition of serum to these quiescent cells, proliferation is reinstated by a series of intracellular events which culminates in the synthesis of DNA followed by mitosis. We propose initially to determine if a cytoplasmic factor can be introduced into quiescent cells which will initiate this progression toward S phase. Our unpublished preliminary data show that human diploid fibroblast recipient cells can be stimulated to enter S phase by fusion with cytoplasts from enucleated lymphoid donor cells which have been synchronized at the G1/S boundary. The time after fusion at which recipient cells enter S phase should indicate whether we have transferred the cytoplasmic factor responsible for the initial transition from the quiescent state or whether we have transferred other factors responsible for the immediate initiation of DNA synthesis. Should the latter be the case, we will use cytoplast donor cells which are synchronized at other phases of the cell cycle or human diploid fibroblasts stimulated from quiescence by serum. Human red cells loaded with unfractionated cytoplast extracts will be fused with recipient cells to determine whether red cells can act as a vehicle for transfer of a cytoplasmic factor. The factor or factors responsible for stimulation will then be isolated by fractionation of extracts from cytoplasts and will be assayed by red cell-mediated microinjection. Finally, the mode of action of the isolated factor will be investigated.